Diagnosis of acute disease is often based on abnormal levels of disease markers, such as enzymes and hormones in biological fluids such as serum, particularly when the concentration changes quickly during the acute phase of disease. For example, the enzyme creatine kinase (CK, ATP:creatine N-phospho-transferase) catalyzes the reversible transfer of a phosphate group from ATP to creatine. It exists as a dimer composed of two subunits commonly identified as the M-subunit and the B-subunit. CK-MB is associated with acute myocardial infarction, and is present in serum in only trace concentrations in the absence of such an episode. Appearance of CK-MB isoenzyme in serum is therefor indicative of myocardial infarction. CK-MM isoforms are present in the serum of normal patients in measurable amounts, but are present in significantly increased concentration following acute myocardial infarction. Assays to determine the occurrence of an acute myocardial infarction by measuring CK isoenzymes are known.
The biological activity and physical properties of proteins such as enzymes and hormones are determined by structural features of the molecule. These features are often modified by endogenous conversion factors present in body fluids. Such conversion may or may not cause the loss of biological activity, or changes in physical properties such as electrophoretic mobility of the molecule. The conversion products may coexist with the original molecule immediately following the onset of an acute disease, but with the passage of time, one finds only the altered protein in the body fluids.
Many tests have been developed which immunologically measure a protein marker in a body fluid. Such immunoassays are often not selective in differentiating native forms of the analyte from altered forms of the analyte. For example, both native and altered forms of CK-MM are immunologically measured using anti-(CK-MM) antibody. Bioassay techniques have been traditionally used to measure enzyme activity. When the altered form of the enzyme marker is inactive, the measurement of enzyme activity provides an adequate measure of changes which occur in the level of the active enzyme in the system. Immunoassays, while offering a more convenient approach, are dependent upon having antibodies which bind selectively with the moiety to be measured. When the altered protein product differs only slightly from a native marker protein, antibodies may be unable to distinguish between the native form and the altered form and will react with both moieties, giving an erroneous result. Immunoassay efforts have generally addressed development of antibodies which bind specifically with the native protein marker together with its altered forms, antibodies binding with only the derivative forms generally being avoided.